Pepstatin A, an inhibitor of acid proteases, inhibited PE-induced lymphoproliferation, whereas leupeptin, an inhibitor of serine and thiol proteases, had no effect ...
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Vol. 60, No. 4
0019-9567/92/041273-06$02.00/0 Copyright ©D 1992, American Society for Microbiology
Lymphoproliferative Activity of Pseudomonas Exotoxin A Is Dependent on Intracellular Processing and Is Associated with the Carboxyl-Terminal Portion PAUL K. LEGAARD, ROY D. LEGRAND, AND MICHAEL L. MISFELDT* Department of Molecular Microbiology and Immunology, University of Missouri-Columbia, School of Medicine, Columbia, Missouri 65212
Received 5 August 1991/Accepted 7 January 1992
Pseudomonas aeruginosa exotoxin A (PE) represents a microbial superantigen that requires processing by accessory cells in order to induce the proliferation of V38-bearing murine T lymphocytes. In this study, we have observed that PE requires intracellular processing by a protease in order to induce lymphoproliferation. Pepstatin A, an inhibitor of acid proteases, inhibited PE-induced lymphoproliferation, whereas leupeptin, an inhibitor of serine and thiol proteases, had no effect on PE-induced lymphoproliferation. A number of mutant forms of PE were examined for their ability to induce lymphoproliferation. The mutant form which lacks amino acids 5 to 224 of the receptor-binding domain, PE43, was capable of inducing murine thymocytes to proliferate in the presence of accessory cells. However, neither PEgly276, a mutant toxin which undergoes a different intracellular processing pattern than wild-type PE, nor PE589, a mutant toxin which lacks amino acids 590 to 613 at the carboxyl terminus, was able to induce thymocyte proliferation. In addition, the lymphoproliferation induced by the PE43 mutant form of PE could also be inhibited by pepstatin A. Therefore, our data indicate that intracellular processing by a proteolytic enzyme which is inhibited by pepstatin A is critical for PE-induced lymphoproliferation. Furthermore, the lymphoproliferative activity of PE is associated with the carboxylterminal portion of PE. The term superantigen has been proposed to describe a number of bacterial toxins that form ligands with major histocompatibility complex (MHC) class II molecules that are able to stimulate the proliferation of a large number of T lymphocytes (42). These superantigens have certain common properties. Superantigens require accessory cells (AC) that bear MHC class II molecules to stimulate T-lymphocyte proliferation. For example, staphylococcal enterotoxins A and B and toxic shock syndrome toxin 1 require presentation by AC expressing MHC class II molecules to stimulate T lymphocytes (8, 9, 18). In addition, antibodies to MHC class II molecules have been observed to block the proliferation of murine T lymphocytes induced by these superantigens (40, 44). Another prominent feature of superantigens is their ability to stimulate T lymphocytes via the V. element of the T-cell receptor. The superantigen staphylococcal enterotoxin B stimulates murine T lymphocytes expressing the V^ 7, 8.1, 8.2, and 8.3 elements within their T-cell receptor (7, 22, 42) and human T lymphocytes expressing the V. 3, 12, 14, 15, 17, and 20 elements (12, 25). Finally, in contrast to conventional protein antigens, superantigens do not require processing prior to their presentation in order to induce T-cell proliferation. Pseudomonas aeruginosa exotoxin A (PE) exhibits a number of properties that are similar to those of other microbial superantigens. First, PE requires AC for the induction of murine thymocyte proliferation (28). Second, antibodies to MHC class II molecules block the thymocyte proliferation induced by PE (27). In addition, PE stimulates murine thymocytes that express the V38 element within their T-cell receptor (27). However, in contrast to the other microbial superantigens, PE requires processing by AC in
*
order to
cause thymocyte proliferation (27). Another superantigen, Mycoplasma arthritidis supernatant, has also been reported to require AC for processing as well as presentation to activate T lymphocytes (5). Thus, some microbial superantigens may require only presentation by AC to stimulate T lymphocytes, whereas other microbial superantigens may require processing and presentation by AC. The participation of AC in immunological reactions has been well established (39). Macrophages have been observed to participate in T-lymphocyte-mediated immunity to exogenous protein antigens by processing the antigen, presenting the antigen to T lymphocytes in the context of MHC class II molecules, and releasing costimulatory molecules. The activation of the T lymphocyte is a result of the interaction of the peptide-MHC class II complex with the T-cell receptor (26, 33, 34). The forms of protein antigen that can associate with MHC class II molecules subsequent to processing by AC include native molecules, unfolded polypeptide chains, and proteolytically derived fragments (1). A number of antigens require limited proteolysis by AC in order to stimulate T-lymphocyte proliferation (13, 36, 39, 41). Bioactive peptides have been reported to be generated as a result of endosomal protease activity in macrophages (15, 16). Antipain, an inhibitor of thiol and serine proteases, was observed to suppress the proliferation of hen egg lysozyme-restricted T-cell clones (30). Pepstatin A, an inhibitor of acid proteases, and leupeptin, an inhibitor of serine
and thiol proteases, have also been observed to inhibit the processing of certain antigens (6, 30, 37, 38). Therefore, endosomal and/or lysosomal protease activity may be required to process protein antigens in order for antigenspecific T cells to become stimulated. In this study, we have examined the role of intracellular proteases in the processing of PE which results in lymphoproliferation. In addition, we have examined a number of
Corresponding author. 1273
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mutant forms of PE for their lymphoproliferative activity. The data presented in this article indicate that PE is processed by a pepstatin A-sensitive acid protease and that a fragment(s) which contains the lymphoproliferative activity is associated with the carboxyl-terminal portion of PE.
MATERUILS AND METHODS Mice. NFR/NUmm (H-2q) mice were derived from brother and sister breeding pairs maintained in the animal care facilities at the University of Missouri-Columbia School of Medicine from breeding stock originally obtained from Carl Hansen, National Institutes of Health. Mice were maintained in autoclaved microisolator cages containing autoclaved bedding and were fed autoclaved mouse chow and acidified water ad libitum. Mice were serologically monitored for antibody titers against mouse pathogens by the University of Missouri-Columbia Research Animal Diagnostic and Investigative Laboratory. Reagents. PE was purchased from the Swiss Serum and Vaccine Institute, Bern, Switzerland. PE43, PEgly276, and PE589 were generously provided by D. FitzGerald and I. Pastan, National Institutes of Health, Bethesda, Md. Leupeptin, pepstatin A, cathepsin D, peanut agglutinin (PNA), and D-galactose were purchased from Sigma Chemical Co., St. Louis, Mo. Paraformaldehyde (PCHO) was purchased from Fisher Scientific, Pittsburgh, Pa. Pepstatin A was dissolved in dimethyl sulfoxide and stored at -70°C. Culture medium. All thymocyte and peritoneal exudate cell (PEC) cultures were carried out in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hazelton Research Products, Inc., Lenexa, Kans.), L-glutamine, nonessential amino acids, sodium pyruvate, 2-mercaptoethanol, and gentamicin (complete medium). Cell populations. Thymocytes were harvested from 6- to 9-week-old mice. The mice were killed, and an incision was made with scissors up the sternum, exposing the thoracic cavity. Thymuses were removed, and the cells were teased into single-cell suspensions with a cell selector (Bellco Glass, Inc., Vineland, N.J.). Thymocytes were fractionated into immature and mature cell populations by selective agglutination with PNA (31). Briefly, the thymocyte suspension was adjusted to 8 x 108 cells per ml, washed twice with phosphate-buffered saline (PBS), and suspended in RPMI 1640 medium. Equal volumes of PNA (1 mg/ml in PBS) and thymocytes were mixed and incubated for 20 min at room temperature. The cell suspension was gently layered onto PBS-20% fetal bovine serum and incubated for 30 min at room temperature. The immature PNA+ thymocytes, which agglutinated with PNA, sedimented to the bottom of the tube, and the mature PNA- thymocytes remained unagglutinated and were located at the top. The mature PNAthymocytes were collected and washed once with 0.2 M D-galactose and twice with PBS prior to suspension in complete medium. PEC were harvested from each mouse by injecting 10 to 12 ml of RPMI 1640 medium into the peritoneal cavity, massaging the abdomen, and removing the fluid. Erythrocytes were lysed, and PEC were washed several times with RPMI 1640 medium and suspended in complete medium. Proliferative-response cultures. PEC were cultured at a density of 2 x 104 cells per well in a 96-well cluster plate (Costar, Cambridge, Mass.) in 0.2 ml of complete medium. PEC were allowed to adhere overnight at 37°C in a 5% CO2 atmosphere and 100% relative humidity. The plastic-nonadherent PEC were removed and discarded, and 0.1 ml of
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complete medium was added to the plastic-adherent PEC. PNA- thymocytes were added at a density of 106 cells per
well in 0.1 ml of complete medium. Cultures were stimulated as indicated and incubated at 37°C, 5% CO2, and 100% relative humidity for 60 h, at which time 1 ,uCi of [3H]thymidine ([3H]TdR) was added to each well. The cells were collected with a cell harvester (Skatron, Inc., Sterling, Va.) after an additional 12 h of incubation. [3HJTdR incorporation was determined by immersing the glass fiber disks in CytoScint (ICN Biomedicals, Irvine, Calif.) and counting in a 1900 CA liquid scintillation analyzer (Packard Instrument Co., Inc., Chicago, Ill.). The stimulation index represents the mean counts per minute (cpm) of [3H]TdR incorporation by thymocytes cultured with plastic-adherent PEC and PE or a mutant form of PE divided by the mean cpm of [3H]TdR incorporation by thymocytes cultured with plastic-adherent PEC and medium. For proliferative-response cultures containing protease inhibitors, irradiated PEC were seeded at a density of 2 x 105 cells per well in a 48-well cluster plate in 0.5 ml of complete medium. PEC were stimulated by the indicated species of PE in the presence or absence of the indicated concentration of the protease inhibitor. An equivalent amount of dimethyl sulfoxide was added to each well that did not contain pepstatin A to control for the solvent. After 6 or 12 h of incubation, PEC were washed with RPMI 1640 medium and treated with 0.5% PCHO for 30 min at room temperature. The plastic-adherent PEC were washed with RPMI 1640 medium and 0.15 M glycine, and 0.4 ml of complete medium was added to the wells. Freshly harvested PNA- thymocytes were added at a density of 5 x 106 cells per well in 0.1 ml of complete medium. Cultures were incubated and [3HJTdR incorporation was determined as described above.
RESULTS Pepstatin A inhibits PE-induced lymphoproliferation. PE had previously been shown to require processing by MHC class II-bearing AC in order to induce thymocyte proliferation (27). AC stimulated by PE prior to treatment with PCHO were able to induce thymocytes to proliferate, whereas AC fixed with PCHO prior to PE stimulation were unable to induce lymphoproliferation (27). In addition, AC treated with lysosomotropic agents, such as ammonium chloride and chloroquine, were also unable to support the proliferation of thymocytes induced by PE (27). These results suggested that PE required processing by AC in order to induce thymocyte proliferation. To determine whether specific proteases were involved in the processing of PE by AC, AC were cultured in the presence of leupeptin, an inhibitor of serine and thiol proteases (2, 35), or pepstatin A, an inhibitor of acid proteases (2, 4, 35), during stimulation by PE. PNA- thymocytes were added to the AC, and thymocyte proliferation was examined. As shown in Fig. 1, leupeptin did not inhibit thymocyte proliferation induced by PE. In contrast, treatment of AC with pepstatin A inhibited the thymocyte proliferation induced by PE. Pepstatin A inhibited PE-induced lymphoproliferation by 20% at 0.1 mM, 37% at 0.25 mM, and 56% at 0.5 mM. Complete abrogation of thymocyte proliferation was observed in the presence of pepstatin A at doses greater than 0.5 mM. However, these concentrations were also observed to be toxic to AC (unpublished observations). Thus, these results indicated that PE required processing by a pepstatin A-sensitive protease in order to induce lymphoproliferation.
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50
40
z 0
o0 0 z
-
30
ET
20
INTRACELLULAR PROCESSING OF PSEUDOMONAS EXOTOXIN A
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TABLE 1. Thymocyte-proliferative activity of mutant PE forms
IIliE m PE
ea PE + IflPWfl P T+s g PE. P
A
10
LA
0
0.25
Xsei
X.
0.5 1.0 0.1 PROTEASE INHIBiTOR (mM)
0.25
0.5
FIG. 1. Pepstatin A inhibits PE-induced lymphoproliferation. PEC were cultured in medium with 50 ng of PE in the absence of protease inhibitors or in the presence of 0.25, 0.5, or 1.0 mM leupeptin or 0.1, 0.25, or 0.5 mM pepstatin A for 6 h, treated with 0.5% PCHO, washed, and added to PNA- thymocytes. The data are expressed as the mean cpm + standard deviation and are representative of a minimum of three experiments. For comparison, PNA- thymocytes cultured in medium gave 567 + 58 cpm, and fixed PEC and PNA- thymocytes cultured in medium gave 533 + 117 cpm.
Examination of the lymphoproliferative activity of mutant PE species. The observation that PE required processing by AC to induce lymphoproliferation (27) (Fig. 1) suggested that a fragment(s) of PE could contain the determinant for the lymphoproliferative activity. Therefore, we examined a number of mutant forms of PE for their ability to induce lymphoproliferation and compared their activity with the lymphoproliferation induced by wild-type PE. PNA- thymocytes in the presence of PEC were cultured with various amounts of the mutant forms of PE. The data shown in Table 1 indicate that wild-type PE induced thymocyte proliferation at concentrations as low as 2 ng per well. Maximum [3HJTdR incorporation by PNA- thymocytes stimulated by wild-type PE was observed with a dose of 10 ng per well. PE43, a mutant toxin in which amino acids 5 to 224 have been deleted (11) (Fig. 2), retained the thymocyte-proliferative activity when cultured with AC (Table 1). However, a dose of 2 or 10 ng of wild-type PE was more effective in inducing thymocyte proliferation than PE43 was. Two nanograms of PE43 was approximately 14-fold less potent in its ability to induce lymphoproliferation than 2 ng of wild-type PE. The ability of PE43 to induce thymocyte proliferation was enhanced when higher concentrations of PE43 were added to the cultures. Twenty nanograms of PE43 induced an amount of [3H]TdR incorporation by thymocytes similar to that induced by 10 ng of wild-type PE. The ability of PE43 to induce thymocyte proliferation suggested that the lymphoproliferative activity of PE could be associated with amino acids 225 to 613. PEgly276, a mutant form of PE which undergoes a different intracellular processing pattern than wild-type PE (24) (Fig. 2), and PE589, a mutant toxin which lacks the carboxyl-terminal 24 amino acids (10) (Fig. 2), were also examined for lymphoproliferative activity. As the data show, both mutant toxins were unable to induce thymocyte proliferation (Table 1). Therefore, the results obtained from these experiments indicate that intracellular processing may be critical for the
Thymocyte (mean ['HJTdRproliferationb incorporation [cpm] + SD)
Addition to culturea (ng)
Additioeangto
None Wild-type PE 2 10 20 PE43 2 10 20 PEgly276 2 10 20 PE589 2 10 20
1,408 ± 282 16,706 ± 1,488 (11.9) 21,652 ± 2,516 (15.4) 15,447 ± 554 (11.0) 1,202 ± 542 (0.9) 17,282 ± 2,560 (12.0) 21,977 ± 1,209 (15.6) 1,606 ± 557 (1.1) 1,459 ± 260 (1.0) 1,319 ± 291 (0.9) 1,414 ± 217 (1.0) 1,448 ± 404 (1.0) 1,204 ± 169 (0.9)
a Cultures consisted of plastic-adherent PEC and PNA- thymocytes in a 96-well cluster plate as described in Materials and Methods. Cultures were stimulated with the indicated form of PE. b Results are expressed as the mean [3HJTdR incorporation in triplicate wells and are representative of at least three experiments. The value in parentheses represents the stimulation index, calculated as described in Materials and Methods.
generation of lymphoproliferative activity and that lymphoproliferative activity may be associated with the carboxylterminal portion of PE. Pepstatin A also inhibits PE43-induced lymphoprolifera1
I
0
5
U
3641
III
613
_~~~Vflll U u
PE
A/ -1PE43 224
R-*G 276
-MEE
-I
I
U
1
U
////M
PEgIy276
PE589
_
UH 590A
613
FIG. 2. Protein maps of wild-type PE, PE43, PEgly276, and PE589. The positions of the cysteine disulfide bonds are indicated for wild-type PE and the mutant PE species. Also shown are the functional domains of PE, Ia, Ib, II, and III. PE43 is a mutant toxin that is deleted for amino acids 5 to 224 within domain Ia (9). PEgly276, in which the arginine residue at position 276 has been changed to a glycine residue, is defective in intracellular processing (24). PE589 is a mutant toxin that is deleted for amino acids 590 to 613 at the carboxyl terminus (10).
LEGAARD ET AL.
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z
12
oCL
0.
0
z
3
0
0.25
0.5
PROTEASE
1.0
0.1
0.25
0.5
INHIBITOR (mM)
FIG. 3. Pepstatin A inhibits PE43-induced lymphoproliferation. PEC were cultured in medium with 50 ng of PE43 in the absence of protease inhibitors or in the presence of 0.25, 0.5, or 1.0 mM leupeptin or 0.1, 0.25, or 0.5 mM pepstatin A for 12 h, treated with 0.5% PCHO, washed, and added to PNA- thymocytes. The data are expressed as the mean cpm + standard deviation and are representative of a minimum of three experiments. For comparison, PNA- thymocytes cultured in medium gave 661 + 103 cpm, and fixed PEC and PNA- thymocytes cultured in medium gave 1,002 22
cpm.
tion. Since wild-type PE was observed to require processing a pepstatin A-sensitive protease to exert its lymphoproliferative activity (Fig. 1), we examined whether PE43induced lymphoproliferation was also inhibited by pepstatin A. AC were cultured in the presence of leupeptin or pepstatin A during stimulation by PE43 prior to addition of the PNA- thymocytes. As shown in Fig. 3, leupeptin had no effect on the PE43-induced lymphoproliferation. In contrast, treatment of AC with pepstatin A during the stimulation with PE43 inhibited thymocyte proliferation. The inhibition observed was 20% at 0.1 mM, 45% at 0.25 mM, and 85% at 0.5 mM. Thus, these data indicate that, as previously observed with wild-type PE (Fig. 1), a pepstatin A-sensitive protease is also required for the induction of murine thymocyte
by
proliferation by PE43. DISCUSSION
PE represents a microbial superantigen that induces the proliferation of murine T lymphocytes which express the V.8 element within their T-cell receptor (27). However, in contrast to other microbial superantigens, PE requires processing by AC prior to presentation in order to induce thymocyte proliferation (27). In this report, the participation of an intracellular protease in the processing of PE by AC is examined. In addition, a number of mutant forms of PE were examined for their lymphoproliferative activity in order to locate the region of PE which is responsible for the lymphoproliferative activity. Limited proteolysis of protein antigens following internalization is thought to be a major step in the processing of antigens for presentation to T lymphocytes (39). Proteolytic enzymes, or endopeptidases, are often categorized into four groups, thiol, serine, acid or aspartyl, and metalloproteases (20). Specific proteolytic inhibitors can be used to examine the role of the various proteolytic enzymes in the intracellular processing of antigens. Myoglobin has been observed to require processing by a thiol protease, such as cathepsin
B, in order to be presented and activate myoglobin-specific T-cell clones (38). It has also been observed that the presentation of myoglobin can be inhibited by leupeptin, an inhibitor of thiol and serine proteases (38). In contrast, the processing of mannosylated bovine serum albumin was shown to be inhibited by pepstatin A (16, 43), an inhibitor of acid proteases such as cathepsin D. These proteases appear to be located in an intracellular acidic compartment (16), such as the endosomes (14, 16, 32). Recently, proteolytic activity was identified in early endosomal or prelysosomal compartments by following the digestion of several proteins internalized by receptor-mediated endocytosis (16). Since other protein antigens require intracellular processing and since our previous data indicated that PE required processing by AC, we examined the role of intracellular proteases in the processing of PE which results in lymphoproliferation. The roles of proteases in the processing of PE and subsequent induction of lymphoproliferation were examined by culturing AC stimulated by PE in the presence of specific protease inhibitors. Leupeptin, an inhibitor of some serine and most thiol proteases, did not abrogate the PE-induced lymphoproliferation (Fig. 1). In contrast, pepstatin A, an inhibitor of acid proteases, inhibited the PE-induced lymphoproliferation (Fig. 1). Similar results were -obtained for the -PE43-induced lymphoproliferation (Fig. 3). Therefore, these data suggest that an acid protease is required to proteolytically cleave PE so that AC can present PE, resulting in thymocyte proliferation. One acid protease, cathepsin D, has been reported to be present in all vertebrate species and is the major lysosomal acid protease in human cells (3). Cathepsin D has also been identified in the endosomes of macrophages (14). Our results suggest that an acid protease, which may be cathepsin D, could function to process PE within the AC, resulting in a fragment(s) that induces thymocyte proliferation. Since an acid protease was involved in the processing, we attempted to generate an active fragment(s) of PE by treating the PE with cathepsin D in vitro. However, the cathepsin D-treated PE was unable to stimulate lymphoproliferation when presented by fixed AC (data not shown). One explanation for this finding is that other proteases besides cathepsin D are involved in generating the active fragment(s). Experiments to address this point are ongoing in our laboratory. Our observation that PE required processing by AC to induce lymphoproliferation (27), (Fig. 1) suggested that a fragment of PE could be responsible for the lymphoproliferative activity. Therefore, a number of mutant forms of PE (Fig. 2) were examined for their ability to induce thymocyte proliferation. PE43, which contains a deletion of amino acids 5 to 224, which includes virtually all of domain Ia (Fig. 2), still has the ability to induce thymocyte proliferation (Table 1). The ability of PE43 to cause lymphoproliferative activity suggests that the fragment(s) generated by processing within AC, which contains the lymphoproliferative-activity determinant, must reside within amino acids 225 to 613 of PE. The requirement for higher concentrations of PE43 to induce thymocyte proliferation quantitatively equivalent to that induced by wild-type PE could be the result of the deletion of a majority of the receptor-binding domain of the toxin. Domain Ia, amino acids 1 to 252, has been shown to be responsible for the receptor-binding activity of the toxin (19, 21, 23). Therefore, PE43, which lacks a majority of this region, would be unable to be taken into the cell by receptormediated endocytosis and would require a higher concentration to induce thymocyte proliferation quantitatively equivalent to that induced by wild-type PE. The importance of
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domain I in the binding of PE to its receptor has been demonstrated by the construction of a mutant in which lysine 57 was changed to glutamic acid, which resulted in reduced cell-binding activity (23). Thus, the lack of lysine 57 in PE43 would dramatically reduce the cell-binding activity of this mutant. The lack of lymphoproliferative activity of PE589 (Table 1), the mutant form which lacks the carboxylterminal 24 amino acids (Fig. 2), suggests that this region is critical for thymocyte proliferation. In addition, the inability of PE589 and PEgly276, both of which contain an intact cell-binding domain, to induce lymphoproliferation also argues against the location of the active fragment(s) within domain Ia. Furthermore, PEgly276 has also been reported to have cell-binding and internalization characteristics similar to those of wild-type PE (24). Therefore, our experiments indicate that the fragment(s) generated by the processing of PE within AC which determines the lymphoproliferative activity is located within amino acids 225 to 613, and the carboxyl-terminal 24 amino acids appear to be critical for thymocyte-proliferative activity. Recently, Ogata et al. have analyzed the intracellular fate of PE in mammalian cells (29). Using radiolabeled PE, they demonstrated that proteolytic cleavage was the first step in the processing of PE. Two major fragments were produced, an amino-terminal 28-kDa fragment and a carboxyl-terminal 37-kDa fragment (29). The presence of arginines at positions 276 and 279 within the disulfide loop created by cysteines 265 and 287 was critical for the production of these two fragments. Substitution of glycine for arginine at position 276 (PEgly276) resulted in a mutant PE molecule that differed in its intracellular processing pattern (20). As shown in Table 1, PEgly276 did not induce thymocyte proliferation. Thus, our results suggest that processing of PE to the 37-kDa and 28-kDa fragments may be required for murine thymocyte proliferation. Although the nature of the cell-associated protease which cleaves PE into the 37-kDa and 28-kDa fragments has not been determined, Ogata et al. have suggested that the protease appears to be associated with early endocytic vesicles (29). Furthermore, since many mammalian cells are susceptible to PE-mediated toxicity, this protease may be widely distributed (29). Our data suggest that PE must undergo proteolytic cleavage in order to induce lymphoproliferation, and since wild-type PE and PE43 cannot induce thymocyte proliferation in the presence of pepstatin A, intracellular processing must be an absolute requirement for the generation of an active PE fragment(s) which can result in the observed lymphoproliferation. In summary, the data presented in this report indicate that AC process PE and present a fragnent(s) of PE to thymocytes which results in their proliferation. The fragment generated by processing within the AC requires the participation of a pepstatin A-sensitive acid protease. Since PE43, which lacks most of the receptor-binding domain, still causes lymphoproliferative activity and PE589, which lacks the carboxyl-terminal 24 amino acids, does not cause lymphoproliferative activity, these results suggest that the carboxylterminal portion of PE is critical for lymphoproliferation. ACKNOWLEDGMENTS This work was supported by Public Health Service grant AI-19359 from the National Institute of Allergy and Infectious Diseases to M.L.M. P.K.L. was supported by Public Health Service grant T32-AI07279 from the National Institutes of Health. We gratefully acknowledge the gifts of PE43, PEgly276, and PE589 from David FitzGerald and Ira Pastan. We also thank Karen
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Ehlert for her technical assistance in the preparation of the manuscript.
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